Spinal cord injury (SCI) is a debilitating condition resulting in irreversible loss of motor function below the site of injury. The complex pathology of SCI, involving a cascade of secondary events and the formation of inhibitory barriers, prevent regeneration across the lesion site. However, in rare cases of spontaneous locomotor recovery, neurons spared in the white matter around the lesion sprout collaterals that bypass the inhibitory scar and form functional relay circuits. The long-term goal of this research is to understand mechanisms of plasticity in the spinal cord after injury; identifying cell types, biological factor, and pharmaceautical agents that are involved in these mechanisms will aid in the development of clinical interventions to improve locomotor outcomes. Because of their role in central pattern generation, contributing to coordination and rhythm, excitatory glutamatergic ventral interneurons-V0, V2a, and V3- are candidate populations to examine for roles in rewiring events resulting in gain of function. While the distinct developmental transcription factor profiles that define these interneurons are increasingly well defined, a lack of mature identification markers has made study of endogenous populations in adults difficult. Our lab has recently developed protocols to differentiate V2a and V3 interneurons from embryonic stem cells (ESCs). By using recombineering techniques to generate transgenic ESCs, large, pure populations of these interneurons will be available to study therapeutic targets and for cell replacement strategies. Furthermore, the recent availability of transgenic animals allowing us to lineage trace specific interneurons enables study of endogenous responses to SCI. The first aim seeks to generate and characterize transgenic V2a ESCs for in vitro study and for transplantation in animal models of SCI. Using BAC recombineering, puromycin antibiotic resistance or a fluorescent protein will be inserted under the V2a-specific Chx10 gene, generating pure or traceable ESC- derived V2a interneurons when differentiated using established protocols. The second aim seeks to apply a novel in vitro microdevice to study isolated and co-cultured transgenic ESC-derived and primary interneurons from transgenic reporter mice. We hypothesize that the addition of certain biological factors might significantly improve maturation and the formation of functional synapses in interneuron populations compared to others. The third aim seeks to discover the role of endogenous ventral spinal interneurons on regeneration after dorsal hemisection spinal cord injury in transgenic reporting mice by evaluating axon sprouting, reformation of synapses, and correlation of interneuron-specific sprouting to locomotor recovery. Together, these aims develop in vitro and in vivo platforms to determine the role of ventral interneurons in spinal cord rewiring events after SCI.